Thyroid hormones exert critical effects on development, as well as a variety of metabolic pathways. They bind to thyroid hormone receptors (T3Rs), ()closely related to steroid hormone receptors, and control the expression of specific target genes in a ligand-dependent manner. There are two classes of T3Rs, TR and TR, which are encoded on two separate genes. They bind to specific regulatory sequences (thyroid hormone response element (TRE)) usually found in the promoter area of T3 responsive genes. TREs are composed of hexamer half-sites (AGGTCA) with degeneracy in sequence and orientation. In the absence of T3, c-Erb A proteins generally repress basal transcription. In the presence of the hormone, they positively or negatively modulate transcription. In vitro, T3Rs bind to DNA as monomer, homodimer, and heterodimer with members of the nuclear receptor superfamily, such as retinoic acid receptors (RARs)(1, 2) , RXR(3, 4, 5) , vitamin D3 receptor(6) , PPAR(7) , or COUP-Tf(8) . It is generally assumed that the T3R-RXR heterodimer is a major transcription complex, at least through a DR4 TRE, whereas the T3R homodimer is probably not a significant transcription factor(9, 10) .

In addition, as previously shown for ligand-activated glucocorticoid receptors (11) and retinoic acid receptors(12) , liganded T3Rs repress AP-1 activity(13, 14) . Conversely, stimulation of AP-1 activity by TPA or c-Jun overexpression inhibits T3Rs transcriptional activity(14) . It was proposed that a direct physical interaction between the AP-1 complex and T3Rs leads to a subsequent loss of activity through TRE or AP-1 responsive elements(14, 15) . The involvement of a third partner in stabilization of the T3R-AP-1 complex was also postulated(13, 16) . Therefore, although liganded c-Erb A proteins directly regulate T3 target gene expression, they repress transcription of AP-1-regulated genes. This dual pathway might regulate the expression of two different sets of genes respectively involved in cell proliferation and differentiation(13) .

We have previously shown that T3 stimulation of quail myoblast differentiation was enhanced by T3R overexpression(17, 18, 19) . In this work, we compared two T3-regulated mechanisms in QM7 myoblasts and in HeLa cells. We report that QM7 cells do not significantly express RXR, leading to a cell-specific activity of T3R. In contrast to its action in HeLa cells, c-Erb A1 does not inhibit AP-1 activity in QM7 cells, but RXR transfection induced functionality of T3R-AP-1 interactions. Last, whereas RXR does not affect T3R transcriptional activity through a TRE, it induces such an activity through a DR4 TRE. In agreement with these data, RXR expression potentiates the T3 stimulation of myoblast differentiation. These findings suggest a crucial role of RXR for the regulation of cell differentiation through interactions with T3R and AP-1 activity.

EXPERIMENTAL PROCEDURES

Cell Cultures

Plasmids and Reporter Genes

Transient Transfections and CAT Assays

Electrophoretic Mobility Shift Assay (EMSA)

Statistical Analysis

RESULTS

T3R Transcriptional Activity in HeLa Cells and in QM7 Myoblasts

In both cell types, co-transfection of the pRS c-Erb A1 expression vector induced a T3-dependent transcriptional activation of the TRE-glo-CAT construct (up to 10-fold, p < 0.001; Fig. 1, A and B). Therefore T3R displayed a similar transcriptional activity in QM7 myoblasts and in HeLa cells through a TRE.

Figure 1: Differences in c-Erb A1 transcriptional activity assessed in HeLa cells and in QM7 myoblasts when using a TRE or a DR4 TRE. Cells were transfected with 1 µg/dish of the TRE-glo-CAT (A and B) or the DR4-tk-CAT reporter gene (C and D) together with 2 µg of c-Erb A 1 expression vector. T3 (10M) was added in the culture medium when indicated. The results are normalized to -galactosidase activities and expressed as percentages of control cells CAT activity. A and C, HeLa cells. B and D, QM7 myoblasts. Three independent transfection experiments were performed in each case.

Using the DR4-tk-CAT reporter gene, we observed striking differences. A significant induction of CAT activity by liganded c-Erb A1 was recorded in HeLa cells (3-fold induction, p < 0.005; Fig. 1C). In QM7 myoblasts, T3R significantly decreased basal CAT activity in the absence of T3 (p < 0.025). The addition of the hormone abrogated this inhibition but did not induce any stimulation of CAT activity (Fig. 1D). Therefore, as expected, the liganded T3R was able to stimulate the expression of genes under the control of a DR4 TRE in HeLa cells. However, in QM7 myoblasts, T3R acts only as a transcriptional repressor of gene transcription through a DR4 TRE, and T3 abrogates this activity.

In Contrast to RAR , T3R Inhibits AP-1 Activity in HeLa Cells but Not in QM7 Myoblasts

As expected, T3R and RAR activated by their cognate ligands (10M T3; 10M RA) strongly inhibited the TPA-stimulated AP-1 activity in HeLa cells (in both cases: -80%, p < 0.005; Fig. 2A). However, liganded T3R did not repress the TPA-induced AP-1 activity in QM7 cells (Fig. 2B) or in secondary quail myoblasts (Fig. 2C). Similar results were obtained when AP-1 activity was stimulated by chicken c-Jun overexpression using the -73 col-CAT reporter gene in QM7 cells (Fig. 2D).

Figure 2: In contrast to RAR , ligand-activated c-Erb A 1 does not repress AP-1 activity in quail myoblasts. Cells were transfected with 1 µg/dish of the (AP-1)-tk-CAT (A, B, C, and the left panel of D) or the -reporter gene (right panel of D) together with 2 µg of c-Erb A 1 expression vector or 2 µg of RAR expression vector. TPA (50 ng/ml), T3 (10M), or retinoic acid (RA, 10M) were added in the culture medium when indicated. Stimulation of AP-1 activity was also achieved by transfection of 2 µg of c-Jun expression vector (C). The results are normalized to -galactosidase activities and expressed as percentages of TPA- or c-Jun-stimulated activity in control cells. A, HeLa cells. B, C, and D, QM7 myoblasts. Three independent transfection experiments were performed in each case.

However, in QM7 myoblasts the ligand-dependent repression by endogenous or exogenous RAR occurred as observed in HeLa cells (Fig. 2, A and B). Therefore, activated T3R and RAR do not display a similar activity in myoblasts, whereas no difference could be noted in HeLa cells. These results suggest that interactions of these receptors with the AP-1 complex might not be mediated through strictly identical pathways.

Stimulation of AP-1 Activity Differently Affects T3R Transcriptional Activity According to the Cell Type and the TRE

Figure 3: Stimulation of AP-1 activity differently affects c-Erb A1 transcriptional activity according to the cell type and the studied TRE. Cells were transfected with 1 µg/dish of the TRE-glo-CAT (A and B) or the DR4-tk-CAT reporter gene (C and D) together with 2 µg of c-Erb A 1 expression vector. AP-1 activity was stimulated by TPA treatment (50 ng/ml, A, B, C, and D) or transfection of 2 µg of c-Jun expression vector (D). T3 (10M) was added in the culture medium when indicated. The results are normalized to -galactosidase activities and expressed as percentages of control cells CAT activity. A and C, HeLa cells. B and D, QM7 myoblasts. Three independent transfection experiments were performed in each case.

More striking is the observation that in QM7 myoblasts, TPA induced a strong transcriptional activity of ligand-activated T3R (7-fold induction, p < 0.001) when using a DR4-tk-CAT gene reporter (Fig. 3D). Similar results were obtained using c-jun transfection (Fig. 3D). In contrast, using the same reporter gene, TPA treatment inhibited T3R transcriptional activity in HeLa cells, with an efficiency similar to that recorded using a TRE-glo-CAT gene reporter (Fig. 3C, p < 0.005).

These data suggest that TPA influence upon T3R transcriptional activity depends on the TRE and on the cell type. In particular, an elevated AP-1 activity induces transcriptional functionality of liganded T3R through a DR4 TRE in QM7 myoblasts.

A Major T3R-RXR Heterodimer Is Detected in HeLa Cells but Not in QM7 Myoblasts

When T3R overexpressing HeLa cell extracts were incubated with a direct repeat sequence (DR4) probe, three complexes displaying different binding intensities were detected with complex II on the brink of detection (Fig. 4A, lanes 1 and 3). Using a TRE probe (Fig. 4B, lanes 3 and 5), no significant differences were observed in the binding ability of these three complexes. Using extracts of T3R-overexpressing QM7 myoblasts and a DR4 probe, only two fast mobility complexes (II and III) were observed (Fig. 4A, lanes 2 and 4). However, binding of complex III was strongly reduced when using a TRE probe (Fig. 4B, lanes 2 and 4).

Figure 4: C-Erb A 1 binding on DR4 or palindromic TREs differs in quail myoblasts and in HeLa cells. Gel retardation assays were performed using a DR4 (A) or a palindromic TRE (B). 10 µg of protein of whole extracts of c-Erb A- or poly(A)-expressing cells were used in each case. A, DR4 TRE. Lanes 1 and 3, c-Erb A 1 HeLa extracts. Lanes 2 and 4, c-Erb A 1 QM7 extracts. B, palindromic TRE. Lane 1, poly(A) HeLa extracts. Lanes 2 and 4, c-Erb A 1 QM7 extracts. Lanes 3 and 5, c-Erb A 1 HeLa extracts. Lane 6, poly(A) QM7 extracts.

When a 5-fold protein excess of control HeLa cellular extract was mixed with T3R-expressing QM7 cellular extract, binding of the receptor as three complexes was observed (Fig. 5). Formation of complex I was associated with a decrease of complexes II and III (Fig. 5, lane 2). Preincubation with an antibody raised against c-Erb A1 confirmed that T3R was a component of these three complexes (Fig. 5, lane 6). In agreement with this last observation, an excess of cold DR4 was found to compete binding of complexes I, II, and III to the probe (Fig. 5, lane 5). Interestingly, binding of complex I was also efficiently competed by molar excess of cold DR5 and DR1 probes (Fig. 5, lanes 3 and 4). These data suggest that a T3R partner able to bind to DR5 and DR1 responsive elements is expressed in HeLa but not in QM7 cells.

Figure 5: HeLa complementation restores a c-Erb A heterodimeric binding in HeLa cells competed by an excess of cold DR1 and DR5 probes. Gel retardation assays were performed using a TRE probe as described in the legend to Fig. 4. Lane 1, c-Erb A 1 QM7 extracts. Lane 2, c-Erb A 1 QM7 extracts (1 vol) + nontransfected HeLa extracts (5 vol). Lane 3, c-Erb A 1 QM7 extracts (1 vol) + nontransfected HeLa extracts (5 vol) + 200 ng cold DR1 oligonucleotide. Lane 4, c-Erb A 1 QM7 extracts (1 vol) + nontransfected HeLa extracts (5 vol) + 200 ng cold DR5 oligonucleotide. Lane 5, c-Erb A 1 QM7 extracts (1 vol) + nontransfected HeLa extracts (5 vol) + 200 ng cold DR4 oligonucleotide. Lane 6, c-Erb A 1 QM7 extracts (1 vol) + nontransfected HeLa extracts (5 vol) + c-Erb A 1 antiserum used at a final 1:5 dilution. Ab, antibody.

When RXR and T3R were co-expressed in QM7 myoblasts, a third complex was detected (Fig. 6, lane 7). It displayed the same mobility as complex I in QM7 expressing T3R extracts mixed with control extracts of HeLa cells (Fig. 6, lanes 7 and 8). In addition, preincubation of cell extracts with an antibody raised against all RXR isoforms suppressed the slow mobility signal (complex I) both in T3R-RXR-expressing myoblasts and in T3R-expressing HeLa cells (Fig. 6, lanes 4 and 5). In addition, binding of complexes II and III was not affected in these two cell types, thus demonstrating absence of RXR in these fast mobility complexes.

Figure 6: Evidence that RXR is the heterodimerization partner of c-Erb A in HeLa cells. Gel retardation assays were performed using a TRE probe as described in the legend to Fig. 4. Lane 1, extracts of c-Erb A 1 overexpressing QM7 + c-Erb A 1 antiserum at a final 1:5 dilution. Lane 2, extracts of c-Erb A1 and RXR overexpressing QM7 + c-Erb A 1 antiserum used at a final 1:5 dilution. Lane 3, c-Erb A 1 QM7 extracts (1 vol) + nontransfected HeLa extracts (5 vol) and c-Erb A 1 antiserum used at a final 1:5 dilution. Lane 4, c-Erb A 1 QM7 extracts + RXR antiserum used at a final 1:5 dilution. Lane 5, c-Erb A 1 HeLa extracts + RXR antiserum used at a final 1:5 dilution. Lane 6, c-Erb A 1 QM7 extracts. Lane 7, extracts of c-Erb A 1- and RXR -overexpressing QM7 cells. Lane 8, c-Erb A 1 QM7 extracts (1 vol) + nontransfected HeLa extracts (5 vol). Ab, antibody.

Because only two RXR isoforms ( and ) are characterized in avian species(24, 30) , expression of theses receptors was assessed by Northern blot in proliferative QM7 myoblasts. Whereas the two transcripts were easily detected in 4.5- and 5.5-day-old quail embryo in agreement with previous data(24, 30) , we failed to detect them in QM7 extracts (Fig. 7), in agreement with our EMSA data.

Figure 7: RXR isoforms are not expressed in proliferating QM7 myoblasts. RXR (5 kilobases) and (2.5 kilobases) RNAs were detected by Northern blot analysis using homologous probe in chicken embryos but not in QM7 cells. Lane 1, total RNA from stage 25 chicken embryos (4.5 days in ovo). Lane 2, total RNA from stage 27 chicken embryos (5.5 days in ovo). Lane 3, total RNA from QM7 cells 48 h after seeding (T3 depleted culture medium). Lane 4, total RNA from QM7 cells 48 h after seeding (0.6 nM T3 supplemented medium). 15 and 30 µg were loaded in lanes 1 and 2 and in lanes 3 and 4, respectively.

RXR Expression Differently Influences T3R Transcriptional Activity According to the Nature of the TRE in QM7 Myoblasts

Figure 8: RXR expression affects c-Erb A transcriptional activity when using a DR4 TRE but not a TRE construct. Cells were transfected with 1 µg/dish of the TRE-glo-CAT (A and B) or the DR4-tk-CAT reporter gene (C and D) together with 2 µg of c-Erb A 1 expression vector. A and C, HeLa cells were transfected with 2 µg of RXR expression vector when indicated. B and D, QM7 myoblasts were stably transfected using the RXR (+RXR) or the corresponding ``empty'' expression vector (control cells). E, RXR expression was assessed by comparison of the activation of a DR1-tk-CAT reporter gene in control and RXR-expressing myoblasts (1 µg of reporter gene was transiently transfected in cells grown without or with 9-cis-RA). T3 (10M) was added in the culture medium when indicated. The results are normalized to -galactosidase activities and expressed as percentages of control cells CAT activity. Three (A, B, C, and D) or five (E) independent transfection experiments were performed.

QM7 myoblasts were transfected with pRS c-RXR and pSV2-neo expression plasmids. Control myoblasts were obtained by co-transfecting pRS poly(A) vector with pSV2-neo plasmid. Stable expression of RXR was tested using a DR1 CAT reporter gene (Fig. 8E). Using a TRE-glo-CAT gene reporter, we observed that RXR expression did not significantly influence the liganded T3R transcriptional activity (Fig. 8B) but restored a transcriptional activity of liganded c-Erb A through a DR4 TRE in QM7 myoblasts (about 4-fold induction, p < 0.005; Fig. 8D). Therefore, this set of data brings evidence that T3R could be fully active through a synthetic TRE in the absence of RXR, whereas the T3R-RXR heterodimer is a major transcription complex on a DR4 TRE.

RXR Expression Restores the Functionality of T3R-AP-1 Interactions

Figure 9: RXR expression induces functionality of AP-1-c-Erb A interactions in QM7 myoblasts. QM7 myoblasts were stably transfected using the RXR (+RXR) or the corresponding empty expression vector (control cells). Thereafter, cells were transfected with 1 µg/dish of the [AP-1]-tk-CAT (A), TRE-glo-CAT (B), or DR4-tk-CAT reporter gene (C) together with 2 µg of c-Erb A 1 expression vector when indicated. TPA (50 ng/ml) and/or T3 (10M) were added in the culture medium when indicated. A, ligand-activated c-Erb A represses AP-1 activity in RXR -expressing myoblasts. B and C, stimulation of AP-1 activity inhibits c-Erb A transcriptional activity in RXR -expressing myoblasts when using a TREglo-CAT (B) or a DR4-tk-CAT reporter gene (C). Three independent experiments were performed. Similar results were obtained when RXR expression was obtained by transient transfections experiments.

Conversely, we demonstrated that RXR expression restored the inhibition of T3R transcriptional activity by AP-1 in QM7 myoblasts, whatever the TRE. Using a TRE, TPA stimulation of AP-1 activity strongly inhibited CAT induction by liganded T3R in RXR -expressing myoblasts (-85%, p < 0.005; Fig. 9B). Similar data were obtained using a DR4 TRE (-55%, p < 0.025; Fig. 9C), but a significant induction of CAT activity remained (3-fold induction, p < 0.01; Fig. 9C), suggesting that RXR expression partly preserved the transcriptional T3R activity through a DR4 TRE, even when AP-1 activity was elevated.

Physiological Consequences

Figure 10: RXR expression strongly enhances the stimulation of QM7 myoblast differentiation induced by T3 in control or c-Erb A 1 overexpressing cells. Connectin expression was assessed by cytoimmunofluorescence 2 days after the induction of differentiation with an antibody raised against connectin and a fluorescein-conjugated antibody raised against mouse immunoglobulins (100). When indicated, 0.6 nM T3 was added in the culture medium. A, control cells. B, control cells + T3. C, T3R-expressing cells. D, T3R-expressing cells + T3. E, RXR -expressing cells. F, RXR -expressing cells + T3. G, T3R + RXR -expressing cells. H, T3R + RXR -expressing cells + T3. These microphotographs are representative of three independent experiments.

DISCUSSION

T3R-RXR Heterodimeric Binding Is Detected on a TRE Sequence in HeLa Cells but Not in Avian Myoblasts

Because an excess of cold DR1 or DR5 probes efficiently competed complex I binding to a TRE, RXR, PPAR, or COUP-Tf I and II, which are able to bind to these response elements(31, 32, 33, 34, 35, 36, 37, 38, 39) , could be possible partners of T3R1 in HeLa cells. In EMSA experiments, using 4RX-1D12 antibody reacting against all RXR isoforms(40) , we identified RXR as the partner of T3R in complex I of HeLa cells. Furthermore, RXR expression induced formation of an additional complex in QM7 myoblasts with the same mobility as complex I.

These results were in line with some previous data suggesting that RXR isoforms are not expressed in proliferating myoblasts(41) . The present study also clearly indicates that RXR isoforms are weakly or not expressed in proliferative quail myoblasts: (i) transcriptional activity of 9-cis-RA from a DR1-tk-CAT reporter gene is not significant (Fig. 7); (ii) we failed to detect any T3R-RXR heterodimers in avian myoblasts (Fig. 6); and (iii) we failed to detect RXR mRNAs in our cell extracts.

QM7 Myoblasts Provide a Useful Experimental Model to Study the Influence of RXR upon T3R Activity

However, T3-activated c-Erb A is devoided of transcriptional activity through a DR4 TRE in the absence of RXR: (i) T3R represses the basal expression level in absence of T3; (ii) the addition of the hormone abrogates this inhibition but does not stimulate transcription; (iii) in cells expressing RXR such as HeLa cells, liganded T3R displays a significant transcriptional activity through a DR4 TRE; and (iv) a similar activity is restored in myoblasts after RXR expression. Therefore these data clearly indicate that the T3R-RXR heterodimer is a major transcription complex on a DR4 TRE.

We report a striking exception to this rule. In QM7 cells, TPA stimulation or c-Jun overexpression induces a strong transcriptional activity to liganded T3R through a DR4 TRE. Disruption by T3 of the c-Erb A homodimer binding to a DR4 TRE (10, 42) probably explains the inability of T3R to increase gene transcription by itself. Therefore, it could be proposed that c-Jun acts by stabilizating homodimer binding to DNA. In addition, according to the hypothesis of Pfahl(16) , Jun could function as a bridging molecule between c-Erb A and the transcriptional machinery. However, because we have not observed a similar T3R-Jun interaction in HeLa cells, a muscle-specific protein could be involved in this bridging, as already proposed ( (16) and Fig. 11).

Figure 11: Hypothetic scheme involving AP-1 and RXR in the regulation of myoblast differentiation by T3. This hypothesis only considers results obtained using a DR4 TRE, closely related to natural TREs. It is based on the original proposition of Pfahl(16) . A, in proliferating myoblasts, a high AP-1 activity is recorded, thus repressing differentiation. In these conditions, according to the hypothesis of this study, c-Jun could function as a bridging molecule between T3R bound to a DR4 TRE and the transcriptional machinery. However, our data demonstrate that c-Jun induces a c-Erb A transcriptional activity in myoblasts but not in HeLa cells. These data suggest that another molecule, which may differ from cell type to cell type, is necessary to stabilize Jun binding to the receptor, in agreement with the proposition of Pfahl(16) . We propose that a muscle-specific factor (MSF) expressed in proliferative myoblasts plays this role. Such a mechanism could induce the activation of a set of genes involved in myogenic differentiation by T3. B, RXR expression induces formation of a T3R-AP-1 inactive complex either through DR4 or TPA responsive elements. Molecules involved in the bridging between the T3R homodimer and the transcriptional machinery could be directly released by RXR (inducing inactivity of the transcriptional complex) or indirectly (as a consequence of a disruption of the interaction of the transcription complex with DNA induced by RXR). Consequently, AP-1 activity is strongly inhibited, thus derepressing terminal differentiation. In these conditions, T3 responsive proteins synthetized in A could induce terminal differentiation. In differentiated cells, AP-1 activity remains depressed; T3-regulated gene expression is activated by RXR/T3R heterodimer. In this scheme, the T3 transcriptional pathway is always functional in relation to c-Jun (proliferation) or RXR (differentiation) expression. In conjunction with T3R (but probably with other nuclear receptors such as RARs), RXR represses AP-1 activity and overcomes the differentiation block.

Furthermore, we have obtained original data establishing a major role of RXR in of T3R-AP-1 functionality. In contrast to RAR , T3R does not repress AP-1 activity in quail myoblasts. In addition, TPA stimulation of endogenous AP-1 activity does not inhibit the ligand-dependent transcriptional activity of T3R in these cells. Interestingly, in contrast to COUP-Tf I, PPAR , or RAR , RXR expression restored functionality of T3R-AP-1 interactions in quail myoblasts. Similar data were obtained with RXR expression, thus suggesting that such an activity is specific of RXR isoforms. Functional interactions between RXR and T3Rs are well documented. However, until this work, it was assumed that RXR interaction with c-Erb A only affected the transcriptional activity of T3R. Our data extend the importance of this interaction to the functionality of T3R-AP-1 interactions. Consequently, they suggest that RXR affects all pathways of T3 action.

RXR Plays a Major Role in the Processes of Cell Differentiation

The physiological relevance of these data is well illustrated by the observation that RXR strongly potentiates the stimulation of differentiation induced by T3 in control or in c-Erb A overexpressing myoblasts. Because myoblast withdrawal from the cell cycle is the first event of terminal differentiation, an anticipated differentiation would probably result in a reduced number of muscle fibers and consequently an important impairment of muscle development. As previously observed, RXR is not expressed before induction of terminal differentiation in murine myoblasts(33) . Our data also indicate that in QM7 cells, RXR is not significantly expressed in proliferating cells. Therefore, RXR absence during the earliest steps of muscle development could provide a protection against such a precocious differentiation.

T3 regulates the expression of a large set of genes, involved in developmental processes and in cell metabolism regulation. A lack of RXR expression in proliferating myoblasts inducing a T3R transcriptional inefficiency would probably severely impair cell metabolism and viability if we consider that direct repeats are the most frequently described TREs. Interestingly, AP-1 activity could restore the T3R transcriptional activity. Therefore, our data indicate that alternative mechanisms are involved in the preservation of T3R activity: first, via AP-1 activity, which also inhibits differentiation, and second via RXR expression, which also inhibits AP-1 activity through a T3R-related mechanism and probably derepresses differentiation (Fig. 11).

FOOTNOTES

*

This work was partly supported by Roussel-Uclaf (France), l'Association Française contre les Myopathies, and l'Association de Recherche sur le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed. Tel.: 33-67-61-22-19; Fax: 33-67-54-56-94.

()

The abbreviations used are: T3R, triiodothyronine receptor; TRE, thyroid hormone response element; RAR, retinoic acid receptor; TPA, 12-O-tetradecanoylphorbol-13-acetate; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; DR, direct repeat sequence; PPAR, peroxisome proliferator-activated receptor; COUP-Tf, chicken ovalbumin upstream promoter-transcription factor.

ACKNOWLEDGEMENTS

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